Cyanine dyes

Abstract

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

What is claimed is: 
     
       1. A compound having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R f , R g , R h , and R i  are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkyl and a bond to a carrier molecule; 
 R c , and R d  are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, phosphoric acid and a bond to a carrier molecule; 
 a and e are independently selected from the integers 0, 1, 2, 3 and 4, with the proviso that at least one member selected from a and e is 2 or greater, such that when a is 2 or greater, each R a  is independently selected, and when e is 2 or greater, each R e  is independently selected;
 wherein when a is 2 or greater, ring A is selected from phenyl substituted at least at the 4- and 6-positions with  a R a , and substituted or unsubstituted napthyl; and when e is 2 or greater, ring E is selected from phenyl substituted at least at the 4- and 6-positions with  e R e , and substituted or unsubstituted naphthyl; 
 
 Q is a member selected from: 
 
       
         
           
           
               
               
           
         
         wherein
 n is selected from the integers from 1 to 3; 
 
         R a  and R e , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O)NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, a bond to a carrier molecule and a ring structure formed by joining a member selected from two R a  moieties together with the atoms they are attached, two R e  moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl 
         wherein
 R 9  is a member selected from OR 10 , and NH(CH 2 ) t OR 11    
 wherein
 R 10  is a member selected from H and substituted or unsubstituted alkyl; 
 t is selected from the integers from 1 to 12; 
 R 11  is a member selected from H, and 
 
 
       
       
         
           
           
               
               
           
         
         
           
             wherein
 u is selected from the integers from 1 to 8; and 
 Y is a nucleobase; and 
 
             R 12  and R 13  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl substituted or unsubstituted heterocycloalkyl and a bond to a carrier molecule, 
           
         
         wherein, when R a , R c , R d , R e , R f , R g , R h , and R i  are selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, one or more of R a , R c , R d , R e , R f , R g , R h , R i  is optionally bound to a carrier molecule. 
       
     
     
       2. The compound according to  claim 1 , said compound having a formula which is a member selected from: 
       
         
           
           
               
               
           
         
       
       wherein
 b and j are independently selected from selected from the integers 0, 1, 2, 3 and 4, such that when b is 2 or greater, each R b  is independently selected, and when j is 2 or greater, each R j  is independently selected; 
 R b  and R j , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O)NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, a bond to a carrier molecule and a ring structure formed by joining a member selected from two R a  moieties together with the atoms they are attached, two R e  moieties together with the atoms they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl 
 wherein
 R 9  is a member selected from OR 10 , and NH(CH 2 ) t OR 11    
 wherein
 R 10  is a member selected from H and substituted or unsubstituted alkyl; 
 t is selected from the integers from 1 to 12; 
 R 11  is a member selected from H, and 
 
 
 
       
         
           
           
               
               
           
         
         
           
             wherein
 u is selected from the integers from 1 to 8; and 
 Y is a nucleobase; and 
 
           
           R 12  and R 13  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl and a bond to a carrier molecule, 
           wherein, when R b , R j , R 9 , R 12  and R 13  are selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl, one or more of R b , R j , R 9 , R 12  and R 13  is optionally bound to a carrier molecule. 
         
       
     
     
       3. The compound according to  claim 1  wherein a member selected from R a , R b , R e  and R j  has the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R 20  and R 21  are members independently selected from H, C(O)R 14 , OR 15 , NR 15 R 16 , CR 15 C(O)R 16 , NR 15 C(O) 2 R 16 , SO 3 H, and C(O)NR 15 R 16 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl 
 wherein
 R 14  is a member selected from H, OR 30 , and substituted or unsubstituted alkyl; 
 R 15 , R 16 , and R 30  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl; and 
 
 R 22  is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. 
 
     
     
       4. The compound according to  claim 3 , wherein at least one of R 20  and R 21  is selected from C(O)NR 26 R 27 , and C(O)OR 30  
 wherein
 R 26  and R 27  are independently members selected from alkylsulfonic acid and heteroalkylsulfonic acid; and 
 R 30  is selected from alkylsulfonic and heteroalkylsulfonic acid. 
 
 
     
     
       5. The compound according to  claim 1  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
       6. The compound according to  claim 5  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
       7. The compound according to  claim 6  having the formula: 
       
         
           
           
               
               
           
         
       
     
     
       8. The compound according to  claim 2  having the formula: 
       
         
           
           
               
               
           
         
       
       wherein
 each R a  and each R e  are members independently selected from C(O)R 29 , OR 32 , NR 32 R 33 , CR 32 C(O)R 33 , NR 32 C(O) 2 R 33 , SO 3 H, and C(O)NR 32 R 33 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl 
 wherein
 R 29  is a member selected from OR 31 , and NH(CH 2 ) t′ OR 30    
 wherein
 R 31  is a member selected from H and substituted or unsubstituted alkyl; 
 t′ is selected from the integers from 1 to 12; 
 R 30  is a member selected from H, and 
 
 
 
       
         
           
           
               
               
           
         
         
           
             wherein
 u′ is selected from the integers from 1 to 8; and 
 Y is a nucleobase; 
 
           
         
         R 32  and R 33  are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl. 
       
     
     
       9. The compound according to  claim 1  wherein R a , R b , and R e  are independently selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       in which
 a' is 0 or 1; 
 x is selected from the integers 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, such that when more than one x is present, the value of each x is independently selected; 
 m is an integer which provides a polyethylene glycol moiety of molecular weight at least about 100 daltons; and 
 R 8  is a member selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl. 
 
     
     
       10. The compound according to  claim 1  wherein two or more of R f , R g , R h , R i , R c  and R d  are: 
       
         
           
           
               
               
           
         
       
       wherein,
 f is selected from the integers from 1 to 10. 
 
     
     
       11. The compound according to  claim 1  wherein each of R f , R g , R h , R i , and R c  is: 
       
         
           
           
               
               
           
         
       
       wherein,
 f is selected from the integers from 1 to 10. 
 
     
     
       12. The compound according to  claim 1  wherein both R c  and R d  are: 
       
         
           
           
               
               
           
         
       
       wherein,
 f is selected from the integers from 1 to 10. 
 
     
     
       13. The compound according to  claim 1  wherein a member selected from R a , R b  R e  and R j  is: 
       
         
           
           
               
               
           
         
       
       wherein
 e' and f' are independently selected from the integers from 1 to 18. 
 
     
     
       14. The compound according to  claim 13  wherein R a  and R e  are each: 
       
         
           
           
               
               
           
         
       
     
     
       15. The compound according to  claim 1  in which two, three, four, five or six of R a , R e , R j , R c , R d , R f , R g , R h , and R i , in any combination, include an independently selected R 11  moiety which is: 
       
         
           
           
               
               
           
         
       
     
     
       16. The compound according to  claim 1  in which two, three, four, five or six of R a , R e , R j , R c , R d , R f , R g , R h , and R i , in any combination, include an independently selected dye moiety or dye linker moiety. 
     
     
       17. The compound according to  claim 1  in which two, three, four, five or six of R a , R e , R j , R c , R d , R f , R g , R h , and R i , in any combination, include an independently selected polyvalent moiety. 
     
     
       18. The compound according to  claim 17  wherein said polyvalent moiety has bound thereto one or more member selected from a dye, a dye-linker moiety, and a nucleotide polyphosphate. 
     
     
       19. A method of monitoring an enzyme reaction, said method comprising:
 (a) forming a reaction mixture by contacting said enzyme with a compound according to  claim 1  wherein said compound is a substrate for said enzyme under conditions sufficient for said enzyme and said compound to react; and 
 (b) monitoring fluorescence of said reaction mixture. 
 
     
     
       20. The method according to  claim 19  wherein said enzyme is a DNA polymerase and said compound comprises a nucleic acid moiety which is said substrate for said enzyme. 
     
     
       21. The method according to  claim 19  wherein said enzyme reaction is template directed DNA synthesis. 
     
     
       22. The method according to  claim 19  wherein said reaction is a component of a single molecule DNA sequencing analysis. 
     
     
       23. The compound according to  claim 1 , wherein any one or more of R a , R e , R f , R g , R h , R i , R c  and R d  is R 40 Z o , wherein
 R 40  is selected from a bond and a linker selected from substituted or unsubstituted alkyl and substituted and unsubstituted heteroalkyl; and 
 Z o  is a member selected from a reactive functional group and a bond to a carrier molecule. 
 
     
     
       24. The compound according to  claim 23 , wherein said linker is substituted or unsubstituted alkyl. 
     
     
       25. The compound according to  claim 23 , wherein Z o  is a member selected from an activated derivative of a carboxyl moiety, a sulfonyl halide, a dienophile, a sulfhydryl and a haloalkyl group. 
     
     
       26. The compound according to  claim 2 , wherein R j  is a member selected from: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
       27. The compound according to  claim 1 , wherein Y is a naturally occuring nucleobase.